Ex vivo regional gene therapy has the potential to solve difficult bone loss problems associated with large bone stock deficiency, abundant scar tissue and limited vascular supply for which at the present time there is no consistently satisfactory solution. We have demonstrated proof of concept the potential clinical utility of this strategey by successfully healing critical sized rodent femoral defects with rat and human bone cells transduced with either an adenoviral vector (Ad- BMP-2) or lentiviral vector (LV-BMP-2) containing the cDNA for BMP-2. In a recent comparison of healing of a bone defect with Ad-BMP-2 versus LV-BMP-2 better quality bone was obtained with the LV-BMP-2 treatment. We hypothesized that the more prolonged BMP expression associated with the lentiviral vector was responsible for the better results with the LV vector and that this increased duration of expression may be necessary to achieve adequate bone repair in the challenging biological enviorments seen in patients. In order for gene therapy to be adopted for clinical use it must be cost-effective, safe and there must be a better understanding of the biology of bone repair with transduced cells in order to successfully manage the wide variability bone loss scenarios seen in humans. The goals for this proposal are to assess the clinical potential of a same day ex vivo approach that can be easily adapted for human use; understand the role of transduced cells and host cells in bone formation and repair and; evaluate the safety of an ex vivo strategy using a lentiviral vector. The research proposed in Specific Aim 1 will compare the efficacy of a same day ev vivo strategy with the standard two step cell harvest and expansion approach. In Specific Aim 2 transgenic mice that express GFP under the control of cell differentiation specific promoters will be used to delineate the role of transduced cells and host cells in the healing of criticial sized bone defects. In Specific Aim 3 the safety of ex vivo gene therapy with a lentiviral vector will be assessed with a biodistribution assay and an evaluation of organ toxicity. These proposed Aims should provide valuable information regarding the feasibility, saftey, and biology of ex vivo gene therapy for bone repair. This data should move us one step closer to implementing this human use. PUBLIC HEALTH RELEVANCE: The ultimate goals of this research proposal are to enhance our knowledge of both the biology of bone repair and regional gene therapy, develop a new cost-effective treatment strategy and assess the safety of this therapy in order to move this technology closer to use in humans. In many cases our present treatment regimens are not adequate to manage difficult bone loss problems. Regional gene therapy has the potential to enhance our ability to treat difficult bone repair problems associated with fracture non-union, revision total joint arthroplasty and spinal fusion.